The long-term objective of the program is to understand the mechanism whereby living things are able to convert the energy of ingested foodstuffs into a form (ATP) that is needed to fuel the energy-requiring processes of life. These insights are fundamental to an understanding of body function in health and disease. The project will examine the molecular mechanism of action of beef heart mitochondrial ATPase (F1), the enzyme that catalyzes the last step in the synthesis of ATP from ADP and Pi. The approach to be followed is based on the premise that the driving force for ATP synthesis in oxidative phosphorylation is the free energy of binding of product ATP and that the major requirement for energy in the process is for the release of product ATP from high-affinity catalytic sites. There are three broad areas of research in the program. The first will be an indepth study of the presence of 3 catalytic sites on the membrane-bound form of F1. The project will attempt to provide answers to the question why a most 2 functional sites are demonstrable on the soluble ATPase whereas 3 sites can now be observed on the membrane-bound form. The second area of activity will be a study of the coupling device, that is, the manner in which the energy store represented by an electrochemical potential gradient of protons is linked to the required changes in binding affinity for substrate and products on F1. The third area of research will focus on the nature of unisite catalysis by F1. Experiments are designed that will look for conformational changes in the protein subsequent to binding of ATP in high affinity-catalytic sites and will study new aspects of the elementary steps in unisite hydrolysis of ATP. The project will make use of ATP analogs, of chemical quench and stop-flow techniques and of established methods of measuring ligand binding in catalytic and noncatalytic adenine nucleotide binding sites.